Achievements and Perspectives in Cloned and Transgenic Cattle Production by Nuclear Transfer: Influence of Cell Type, Epigenetic Status and New Technology

DOI: 10.21451/1984-3143-AR853

Anim. Reprod., v.14, n.4, p.1003-1013, Oct./Dec. 2017

Achievements and perspectives in cloned and transgenic cattle production by nuclear transfer: influence of cell type, epigenetic status and new technology

Alinne Gloria Curcio1,4, Fabiana Fernandes Bressan2,3, Flávio Vieira Meirelles2,3, Angelo José Burla Dias1

1Laboratório de Reprodução e Melhoramento Genético Animal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ, Brasil. 2Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia Alimentar (FZEA), Universidade de São Paulo (USP), Pirassununga, SP, Brasil. 3Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Universidade de São Paulo (USP), São Paulo, SP, Brasil.

Abstract Establishing nuclear transfer (NT) (Wilmut et al., 1997) in conjunction with cell culturing and Genetically modified cattle production is efficient genetic modification methods provides new motivated by many factors, including recombinant strategies for producing transgenic livestock due to the protein production for therapeutic purposes, disease possibility of selecting cells prior their use as a nucleus models and animals presenting improved production donor, ensuring a transgenesis efficiency of near 100%. traits. Nuclear transfer (NT), combined with efficient Initially, nuclear reprogramming by NT was cultivation methods, genetic modification and donor cell reported possible using embryonic cells (blastomeres) selection is important for transgenic cattle production. as nuclear donors for both laboratory and farm animals Studies have found that adult cells (such as fibroblasts (Illmensee and Hoppe, 1981; Willadsen, 1986), and it and cumulus cells, among others) used as nuclear was believed that pluripotent cells were responsible for donors achieved results similar to those of fetal cells, nuclear reprogramming success and proper embryo with the advantages of easier collection and a known development (McGrath and Solter, 1983; Prather et al., genotype/phenotype. However, no consensus has been 1987). This technique limited livestock production reached on the influence of cell type on transgene because deriving and maintaining embryonic stem cell expression levels and post-reprogramming capacity (ES) cultures in vitro is not possible as pluripotent cells after nuclear transfer, and these factors appear to be cultured in vitro are not yet fully characterized or related to epigenetic factors. The development of new reproducible in domestic animals (Brevini et al., 2008; strategies, such as chromatin-modifying agents (CMAs), Nowak-Imialek et al., 2011). The birth of the first farm in vitro generation of induced pluripotent cells (iPS animal from nuclear transfer using in vitro cultured fetal cells) and precise genome editing techniques are being cell lines was reported in 1996 (Campbell et al., 1996), explored and may influence nuclear reprogramming and the birth of Dolly “the sheep” (Wilmut et al., 1997) success for efficiently producing genetically modified revolutionized the reprogramming concept at that time bovine clones. by demonstrating that differentiated adult somatic cells could be nuclear donors to produce cloned animals. Keywords: bovine, cloning, epigenetic, nuclear A short time after Dolly’s achievement, reprogramming, transgenesis. Schnieke et al. 1997, demonstrated the first use of NT to produce the first transgenic animal. They produced a Introduction transgenic sheep that secreted factor IX in its milk (Schnieke et al., 1997). Thereafter, other cell types have Commercial and scientific interest in been used to clone and produce transgenic embryos with transgenic animal production has grown worldwide. different transgene incorporation efficiency rates (Arat This is due to the possibility of using animals to et al., 2001; Feng et al., 2015; Gong et al., 2004). produce recombinant proteins for therapeutic purposes Recently developed precise techniques for (An et al., 2012), develop in vivo gene function models genome editing have enhanced the safety and efficiency to study organogenesis and development (Berg et al., of producing transgenic farm animals desirable in both 2011), understand important diseases and produce agriculture and biomedicine (Petersen and Niemann, animals with improved production characteristics (Wu 2015; Rémy et al., 2010). TALENs (transcription et al., 2015). activator-like effector nucleases) technology has DNA pronuclear injection was the first and allowed genetic modification to become more precise most common method for producing transgenic animal and less time-consuming. More recently, CRISPR for years, but low transgenesis rates limited its use. technology revolutionized the animal transgenic field, Murakami et al. (1999) reported that only 2.9% of bringing several advantages such as more precise bovine embryos produced after DNA pronuclear targeting, fewer off-targets, and faster technology injections were genetically modified, and 70.3% of (Carlson et al., 2012; Menchaca et al., 2016; Lotti et al., those were chimeras. This is due to the random insertion 2017). It is important to highlight, however, that of exogenous DNA into the host cell genome efficient TALEN or CRISPR technology protocols for (Murakami et al., 1999). producing transgenic farm animals rely on NT ______4Corresponding author: [email protected] Received: June 24, 2016 accepted: October 10, 2017 Curcio et al. Enhancing transgenic cattle production. procedures to assure high efficiency in terms of modified individuals, but their use in producing transgenic offspring birth. Therefore, evaluating the cell transgenic farm animals is challenging as was phenotype for the precise genetic modification to be previously described regarding ES cells from domestic performed remains a concern. animals (Blomberg and Telugu, 2012; Gandolfi et al., Although NT uses different cells types, the 2012). A brief summary of important NT achievements influence of the cellular differentiation stage on both is shown in Figure 1. cloning and transgene integration efficiency are of After the NT of a differentiated adult cell interest. This relationship is not well-defined, and more (mammary gland cell) to an enucleated oocyte, the birth studies are needed to fully understand it; however, it is of Dolly the sheep in 1996 confirmed speculation that clear that epigenetic factors are involved, affecting even after having reached a certain stage of primarily post-nuclear transfer reprogramming differentiation, differentiated somatic cells could be efficiency (Smith et al., 2012, 2015). reprogrammed if aided by a cytoplasmic apparatus This review (i) presents a brief history of the (Wilmut et al., 1997). cell types used in cloning and transgenic cattle Therefore, the possibility of using others cell production, (ii) addresses the epigenetic issues that may types was investigated for producing both clones and affect transgenic and cloning cattle production efficiency transgenic cattle by NT. In 1998, Kato et al., reported and (iii) describes current strategies, such as chromatin- the birth of eight heifers produced by nuclear transfer modifying agents (CMA), iPS cells and endonucleases using cumulus cells and oviduct epithelial cells from an as means to innovate and improve results. adult animal (Kato et al., 1998). In 2000, the same group compared clone production efficiency using adult Use of somatic cells by nuclear transfer in transgenic female cells (cumulus, oviduct and uterine cells) and ear cattle production and skin cells from neonates and adult male cattle. These authors observed that clones from adult cells The term “transgenic” refers to an organism often died in the final stages of gestation, and those that whose genome was permanently altered by insertion, survived often had abnormalities (Kato et al., 2000). modification or inactivation of DNA, with the genetic The authors attributed these findings to donor cell modification being transmitted to its offspring (Rülicke mutations and telomere shortening, factors that could et al., 2007). In livestock production, transgenic animals occur in aging animals. Today, it is known that these have been developed primarily for use as bioreactors to modifications are also caused by epigenetic failures in produce high quality medicinal proteins on a large scale, donor cell reprogramming (Yang et al., 2007; Song et with lower costs and higher efficiency compared to al., 2014). other production methods such as bacterial, yeast and Arat et al. (2001) demonstrated the first use of cells culturing (Houdebine, 2009; Bagle et al., 2012). adult cells (granulosa cells) for transgenic embryo NT is the most common method for transgenic production in cattle. Cho et al. (2004) reported that the cattle production (Jeong et al., 2016; Lu et al., 2016; embryo development rate post nuclear transfer, as Ren et al., 2017; Gao et al., 2017;). This is primarily assessed by green fluorescent protein (GFP) expression, due to the possibility of identifying the transgene in the was greater when smaller cumulus cells from early cell genome before using it as a nuclear donor, which passages were used compared to adult and fetal may avoid chimera production, genetic anomalies in the fibroblasts. According to the authors, using small offspring, homogenous offspring production or an cumulus cells allows for better nucleus-cytoplasmic unintentional knock-out due to transgene localization in interaction in the recipient cytoplast. Moreover, when the non-coding DNA (Bressan et al., 2008, 2011). NT is performed with metaphase II oocytes, The NT technique fuses one diploid cell synchronized cells in the G0/G1 phase of the cell cycle (embryonic, fetal or adult), with an enucleated oocyte (smaller cells) are more effective in embryonic (recipient cytoplast) and is chemically activated to development (Campbell, 1999). Concerning passage trigger embryo development. Thus, the recipient oocyte number, prolonged culturing periods can potentially must induce nuclear reprogramming and support result in genetic alterations. Genetic alteration is a major embryonic development, while the donor cell nucleus factor in cloned and transgenic production. It is not fully must be totipotent (Wilmut et al., 2015). understood; however, interestingly, embryos resulting Many cell types and culture and manipulation from NT failed to express some genes related to nuclear conditions (presence or absence of bovine fetal serum, reprogramming (e.g., Oct-4 gene) and placentation cell passage number, oxygen tension and others) have (Yang et al., 2007). been studied for their effects on transgene expression Higher production rates of transgenic bovine levels in the donor cell nucleus and the cloned calves blastocysts were also obtained using cumulus (44.6%) (Cho et al., 2004; Gong et al., 2004; He et al., 2016). and oviduct fetal epithelial cells (49.1%) than with fetal As previously discussed, initial studies used fibroblasts (32.7%) (Gong et al., 2004). This indicates embryonic stem cells from embryos in the morula or that adult cells can efficiently produce transgenic cattle. blastocyst stage for their ability to generate any In some situations, adult cells may be more suitable embryonic tissue (Puri and Nagy, 2012). In mice, than embryonic or fetal cells due to easier collection and embryonic stem cells efficiently produce genetically knowing the genotype/phenotype.

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Figure 1. Timetable of a brief summary of important NT achievements.

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In 2006, Sung et al. tested hematopoietic Changes in the chromatin, such as methylation and mouse cells at different differentiation stages histone modifications, are highly correlated and alter (hematopoietic stem cells (HSC), progenitor cells and gene transcriptional activity by leaving chromatin granulocytes) and found that cloning efficiency exposed or protected from transcription machinery increases with the donor cell’s differentiation state, and (Cheng and Blumenthal, 2010). granulocytes were the best cell type for cloning. The DNA is a long molecule that must be packaged authors concluded that hematopoietic cells appeared to for nuclear containment. Thus, the chromatin is be an exception to the hypothesis that “undifferentiated wrapped around a histone octamer, constituting the cells are more efficient than differentiated somatic cells nucleosome, the DNA’s basic organizational unit. for cloning production”. Inoue et al. (2006) Histones are proteins with a carboxy-terminal domain demonstrated that cloned embryos derived from HSC and amino-terminal tail where epigenetic modifications failed to express five of the six important embryonic occur. The main histone modifications include genes examined, including Hdac1 (encodes histone methylation of lysine and arginine residues, acetylation deacetylase 1), a key zygotic gene activation regulator. of lysine residues, and ubiquitination, sumoylation and These results were attributed to less plasticity of phosphorylation of serine and threonine residues. Of hematopoietic stem cells (Inoue et al., 2006). these, lysine residue acetylation and methylation are the Mammary gland epithelial cell use in most well-known and studied (García et al., 2012). transgenic animal production has grown due to its Histone acetyltransferase (HAT) and histone applicability (Zhan et al., 2017). Regarding recombinant deacetylase (HDAC) are the enzymes that control protein production, Feng et al. (2015) used mammary histone acetylation levels. Histone hyperacetylation is gland cells to produce transgenic sheep expressing the associated with chromatin regions with high alpha-lactoferrin gene. After transfection with plasmid transcriptional activity, while hypoacetylation is found encoding alpha-lactoferrin, cells secreted recombinant in low activity regions (Schmittwolf et al., 2005). protein at detectable levels in the culture media. Histone methylation may be related to non-transcribed Similarly, our group reported generating live offspring or transcribed regions, depending on which residue is after donor cell NT from developing mammary glands methylated. For example, histone H3 lysine residue 9 in cattle. We described constructing specific vectors to methylation (H3K9me) and histone H3 lysine residue encode the B-casein promoter and the hFIX gene, as 27 methylation (H3K27me) are related to non- well as integrating them into cattle donor cells and transcribed regions, while histone H3 lysine residue 4 generating offspring (Monzani et al., 2011, 2013). This (H3K4me), 36 (H3K36me) and 79 (H3K79me) improvement represents another effective method for methylation are related to transcribed regions. These selecting cells prior to their use as nuclear donors and events are controlled by histone methyltransferase analyzing tissue-specific promoter activity. enzyme (HMTs). Fetal and adult fibroblasts remain the most DNA CpG island methylation is a primary common cell type for producing transgenic cattle by epigenetic mechanism that directly affects gene NT. This may be due to the ease of collection and in expression as some transcription factors only bind to vitro culturing and that the cells divide several times unmethylated DNA sequences; thus, methylation may before reaching senescence. In theory, different cell induce gene sequence expression loss (Salozhin et al., types could be used to produce clones and transgenic 2005). Methyltransferases are enzymes that control animals by NT with different success rates, but nuclear DNA methylation levels. DNA (cytosine-5)- reprogramming efficiency appears to be related to donor methyltransferase 1 (DNMT1) maintains methylation cell nucleus plasticity and the capacity to undergo levels during DNA replication. DNMT2 is related to nuclear programming before NT. The role of the RNA methylation to enhance tRNA stability, and nucleus donor cell’s epigenetic status on DNMT3A and DNMT3B are responsible for de novo reprogramming efficiency is thus gaining interest, and methylation (Iager et al., 2008). this relationship will be further discussed below. Proper DNA methylation reprogramming is essential during gametogenesis and early embryogenesis Epigenetic factors related to animal production to generate healthy offspring; therefore, its disruption or incomplete reprogramming may directly affect other Epigenetic modifications are heritable changes processes such as NT. Male and female gametes have a in DNA structure and organization that are unrelated to hypermethylated genome due to gametogenesis, and changes in the nucleotide base sequence. The term was after fertilization, the pro-nuclei undergo a process first proposed to explain how cells carrying identical called "demethylation". In the male pro-nucleus, this is DNA could express different genes (García et al., 2012). a fast and active process, whereas in females, it occurs This concept is now well-recognized, and these slowly and progressively (Yang et al., 2007). At the 8- modifications occur through changes in the chromatin 16 cell stage of concurrent embryonic genome that alter gene transcriptional activity; however, the effect activation, epigenetic parameter reprogramming occurs, of these alterations remains unclear. Herein we will focus known as de novo methylation. These processes are on epigenetic modifications that primarily occur by two usually coordinated, culminating in successful embryo distinct mechanisms: (i) histone modification and (ii) implantation and embryonic and fetal development. cytosine methylation in CpG islands in double-stranded During early embryonic development and cell DNA (Cheng and Blumenthal, 2010; Reik et al., 2001). differentiation, epigenetic markers are gradually

1006 Anim. Reprod., v.14, n.4, p.1003-1013, Oct./Dec. 2017 Curcio et al. Enhancing transgenic cattle production. established, forming different heritable epigenetic Matoba et al. (2014) identified mouse patterns that are important for maintaining the identity reprogramming resistant regions (RRRs) that were well- of differentiated cells (Schmittwolf et al., 2005). expressed in 2-cell in vitro fertilized embryos, but not in In nuclear transfer, nuclear reprogramming cloned embryos. These RRRs are enriched for involves two complex epigenetic steps: (i) H3K9me3 (trimethylation of histone H3 on lysine dedifferentiation of a differentiated somatic cell to a residue 9 H3K9me3) on the donor cell genome. totipotent stage by epigenetic marker removal in the According to these authors, this region is a major barrier donor nucleus and (ii) redifferentiation of totipotent to efficient nuclear reprogramming in mice and its embryonic cells into all tissue types of a new animal removal by ectopically expressed H3K9me3 (Yang et al., 2007). Such events occur quickly, whereas demethylase, Kdm4d, significantly improved SCNT in naturally fertilized embryos, such epigenetic markers efficiency (Matoba et al., 2014). are established during gametogenesis and fertilization, Thus, more recent approaches have focused on and thus may diminish NT efficiency. the possibility that open chromatin configurations Despite current efforts and findings in this (already found in stem cells, embryonic and fetal cells) field, embryo production by NT remains low at could benefit nuclear reprogramming (Song et al., 2014). approximately 1-5%, and embryos derived from NT Because the use of embryonic stem cells in farm animals typically present implantation failures, fetal is still incipient, new strategies have been developed to abnormalities, and gestational, placentation and calving optimize clones and transgenic animals produced by NT. problems. Many of these abnormalities are due to Among these strategies, using chromatin-modifying nuclear reprogramming failure, caused by changes in agents (CMAs) in donor cells prior to NT, as well as the histone parameters, DNA methylation and gene possibility for using non-embryonic but pluripotent expression related to early embryogenesis and stem cells (the induced pluripotent stem cells or iPS placentation (Bressan et al., 2009; Smith et al., 2015; cells) as donor cells have shown significant potential Suzuki et al., 2009; Yang et al., 2007). and are further discussed herein. Several studies report that the more advanced the developmental stage of the nuclear donor cell, the Modeling cellular epigenetic status for nuclear lower the efficiency of the embryo production by NT. In transfer mice, for example, reduced clone production occurs when blastomeres at a more advanced developmental stage are Due to growing evidence of epigenetic used as donor cells (22% - 2 cells, 14% - 4 cells and 8% - reprogramming failures in NT embryos, chromatin- 8 cells). In cattle, NT blastocyst production rates were modifying agents (CMAs) are being studied in several reported at 28% when blastomeres were used as nuclear species such as cattle, sheep, buffalo, mice, swine and donors. When fetal and adult fibroblasts were used, rabbits (Cervera et al., 2009; Hu et al., 2012; Iager et efficiency was significantly reduced (13% and 5%, al., 2008; Kishigami et al., 2006; Luo et al., 2013; Shi respectively) (Wilmut et al., 2002). et al., 2008). CMAs are chemical substances that change Blelloch et al. (2006) demonstrated that stem cellular epigenetic patterns. Trichostatin A (Kishigami et cell production efficiency in mice was higher when al., 2006), sodium butyrate (NaB) (Shi et al., 2003), m- neural stem cells were used as NT donors compared to carboxycinnamic acid bishydroxamide (CBHA) (Song et differentiated neuronal cells. They also demonstrated al., 2014), Scriptaid (Wang et al., 2011) and valproic acid that hypomethylation of differentiated cell genomes (Xu et al., 2012) are examples of substances used for NT increased clone production (Blelloch et al., 2006). procedures, as shown in Figure 2.

Figure 2. (A) Schematic drawing of histone acetyltransferase and histone deacetylase activity. Note that using histone deacetylase inhibitors may improve histone acetylation levels, which could increase chromatin exposure to transcription machinery. (B) Examples of histone deacetylase inhibitors.

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In cattle, use of trichostatin A (TSA), a histone quality compared to the untreated control (CTR-NT) deacetylase inhibitor, led to production of 8-cell cloned revealed few genes were differentially expressed in bovine embryos with histone H4 acetylation levels at TSA-NT embryos (1907 = 5.1% versus CTR-NT 4.3%). lysine 5 (H4K5ac) similar to their fertilized counterparts According to the authors, this suggests that the and significantly greater than the control group. This imperfect expression of a few genes can result in a treatment has also improved in vitro embryo production, defective phenotype due to a ripple effect, which is not reaching levels close to those of in vitro fertilization completely responsive to TSA treatment (Hosseini et (IVF) embryos (Iager et al., 2008). According to Kong al., 2016). et al. (2014), TSA also increases telomere length and Although much has been reported on cloned may be a mechanism by which this substance improves embryo production, minimal and controversial data are cloned embryo development (Kong et al., 2014). available regarding the offspring, especially from In 2003, Shi et al. analyzed the effect of domestic animals. Our group has studied whether treating nucleus donor cells with different chromatin- CMAs targeting chromatin acetylation and DNA modifying agents: Trichostatin A; 5-aza-2- methylation can alter chromatin configuration to deoxycytidine (Aza-C, DNA methylation inhibitor); 5- improve nuclear reprogramming and enhance offspring bromodeoxyuridine; and sodium butyrate (NaB) on results. Thus, bovine fibroblasts were treated with either embryo development. Interestingly, NaB, another 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA), histone deacetylase inhibitor, improved the proportion hydralazine (HH) plus valproic acid (VPA) or VPA of cloned embryos that developed to blastocyst stage alone and used as NT donor cells. Cloned bovine (59%) compared to the untreated group (26%) (Shi et zygotes were also treated with TSA to test whether the al., 2003). effect would be more pronounced in cells or embryos. In swine, the use of CBHA increased blastocyst Although live offspring were born from both the control formation as well as the levels of histone 3 acetylation and treated groups, no evidence that either nuclear at residue 9 (H3K9ac) and residue 18 (H3K18ac) and donor cells or cloned zygotes with CMAs positively histone 4 at residue 16 (H4K16c). It also increased affected pre- and post-implantation development of development-related gene expression, such as POU5F1, cloned cattle (Sangalli et al., 2012, 2014). Thus, more CDX2 and the imprinted IGF2 gene (Song et al., 2014). studies are needed to identify potentially important Valproic acid (VPA) is a fatty acid used to treat deregulated bovine genes to find strategies to correct central nervous system diseases. Studies have shown it their expression and to identify possible targets of induces differentiated cell reprogramming. In miniature disrupted maternal recognition before the embryos are pigs, embryos treated with VPA showed enhanced Oct- transferred to recipients. 3/4 expression and in vitro development after cell nuclear transfer (Miyoshi et al., 2010). In bovines, Xu et iPS cell use for cloned animal production by nuclear al. (2012) tested different VPA concentrations at transfer different time periods for activation. They found that 4 nM of VPA over 24 hours increased cleavage and iPS cells are adult cells that have been blastocyst rates, reduced apoptosis in blastocysts and genetically induced into a pluripotent state similar to improved immunofluorescent signals for H3K9ac in a embryonic stem cell stages by introducing the specific pattern similar to that of in vitro fertilized (IVF) genes, Oct4, Sox2, KLF4 and C-MYC, known as OSKM embryos (Xu et al., 2012). factors (Takahashi and Yamanaka, 2006; Takahashi et Using scriptaid 14 hours after activation al., 2007). The role of other factors, such as Nanog, Lin- increased NT bovine embryo production in vitro and 28, TCL-1A, has also been evaluated (Yu et al., 2007; immunofluorescent signals for H3K9ac, decreased Picanço-Castro et al., 2011). fluorescent signals for H3K9m2 in all analyzed iPS cells are a new type of pluripotent stem cell embryonic stages (two-cell, eight-cell, and blastocyst that are highly proliferative and can form different stages) and increased expression levels of two tissues. iPS cells are similar to embryonic stem cells in developmentally important genes, Interferon tau (IFN-t) morphology, pluripotent gene expression, promoter and Oct4 (Wang et al., 2011). A recent work also methylation level, teratoma formation capacity and demonstrated that Scriptaid increased in vitro ability to differentiate into all cell types (Cao et al., development of NT mini-pig embryos and improved 2012). acetylation levels on H3K14 and development-related In vitro pluripotent cell production technology gene expression (AKT, Oct4 and PGC-1α) (Zhang et al., plays an important and unique role in animal production 2017). because “true” stem cells cannot be efficiently produced Interestingly, less than twenty genes are or maintained in vitro, as previously discussed (Muñoz typically deregulated in these embryos, and it is et al., 2008; Nowak-Imialek et al., 2011). Conversely, important to identify which gene networks are disrupted iPS cells have already been reported in several species, (Beyhan et al., 2007). A recent microarray study used including both domestic (e.g., cattle, dog, sheep, swine, Trichostatin A to evaluate the effects of assisted horses) and endangered animals (e.g., felids) epigenetic modification of NT bovine blastocyst (Gonçalves et al., 2014; Koh and Piedrahita, 2014; transcriptional profiles. Despite TSA treatment (TSA- Ezashi et al., 2016). NT) and improved epigenetic reprogramming iPS cells, as well as embryonic stem cells, parameters, in vitro embryonic development yield and allow accurate genetic manipulation (Park and Telugu,

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2014). In cattle, their use accelerates improvement of to generate a non-homologous end-joining (NHEJ) desirable genetic traits in livestock, which has been the repair pathway. This technique reduced off-target aim of different research groups (Cao et al., 2012). effects and both transgenic bovine fetal fibroblasts and Recently, studies using iPS cells as donor nuclei for NT cattle were efficiently produced (Gao et al., 2017). have been reported in mice (Zhou et al., 2010; Liu et Bevacqua et al. (2016) reported using the CRISPR/Cas9 al., 2012) and swine (Cheng et al., 2012). Although system to induce knockout and knock-in alleles of the these studies have reported that cloned embryos were bovine PRNP gene responsible for mad cow disease, generated after NT using iPS cells, no major both in bovine fetal fibroblasts and in IVF embryos improvement on embryo production was reported until (Bevacqua et al., 2016). now. Therefore, despite the advances that combining The CRISPR-Cas 9 system has also been used these two biotechniques may bring to the reproductive to produce knockout genes. Choi et al. (2015) reported and biomedical fields, more solid and reliable studies disrupting chromosomally integrated exogenous eGFP are soon expected. genes using an RNA-guided endonuclease in bovine transgenic somatic cells. The fibroblasts were efficiently Perspectives in animal transgenesis and conclusions used for NT and developed in the blastocyst stage. This system may also be used for DNA labeling, regulating Efforts are underway in the cloning and gene expression, RNA cleavage, gene mapping and transgenesis fields, as the efficiency of the animals that RNA screening (Hale et al., 2009; Choi et al., 2015; are born remains low. The use of transgenic iPS cells to Deng et al., 2015). Recently, Jeong et al. (2016) enhance TN efficiency is promising but has not reported the first production of pharmaceutical products currently been tested or validated on a large scale. The in cattle using the CRISPR-Cas9 system. They use of CMAs, however largely studied, still presents produced human fibroblast growth factor 2 (FGF2) controversial results regarding its efficiency in transgenic fibroblasts using CRISPR-Cas9-mediated improving offspring number and health. However, homologous recombination. According to the authors, transgenesis techniques have evolved into safer and FGF2 was secreted in culture medium and when used as simpler techniques. Thus, endonucleases appear to have a nucleus donor in NT, the blastocysts produced were a key role in facilitating mammalian genome also transgenic (Jeong et al., 2016). engineering. In brief, precise genome editing technologies Endonucleases are restriction enzymes that are widely used in cattle and other domestic animals to cleave DNA in a specific manner. Meganucleases, zinc improve transgenesis (Menchaca et al., 2016; Niu et al., finger nucleases (ZFNs) and transcription activator-life 2017; Wu et al., 2017), and most of these studies rely effector nucleases (TALENs) were the first three classes on TN technology to produce live offspring. It is of customizable DNA-binding proteins developed, but possible that the knowledge gained through the their low-specificity limits their use (Peng et al., 2016). CRISPR-Cas 9 system may soon be used in cloning Recently, a defense system against foreign production. The system should be used to identify genes nucleic acids, such as viruses or plasmids, initially that do not work well after nuclear reprogramming and described in prokaryotes, has been used to improve to correct the problems associated with the SCNT transgene efficiency (Mojica et al., 2000). The CRISPR technique. (clustered regulatory interspaced short palindromic In conclusion, (i) different cell types can repeats) technology uses repeated short sequences theoretically be used for cloning and transgenic interspaced by non-transcribed regions (spacers) that are production with different rates of nuclear closely associated with gene regions (CRISPR reprogramming success and transgene incorporation. (ii) associated genes – Cas) that codify nucleases, Nuclear reprogramming efficiency appears to be closely polymerases and helicases, which are fundamental to related to the differentiation stage of the donor cell the system’s function (Jansen et al., 2002). nucleus, and its success rate is higher when using less Therefore the CRISPR-Cas 9 system is a differentiated cells in SCNT, such as stem and fetal powerful, precise and relatively simple tool developed cells. (iii) These results are likely due to epigenetic for gene editing, based on the bacterial CRISPR-Cas marks, and (iv) studies on CMAs and iPS cells have defense system (Jinek et al., 2012; Cong et al., 2013). shown promising results in this field. Finally, (v) the use The technique refers to RNA guides (CRISPR) that of RNA-guided endonucleases should improve orient the endonuclease, Cas 9, to cleave the DNA transgene expression in offspring. The knowledge sequence in a specific manner. Using CRISPR-Cas 9 gained in this field could be used to improve nuclear makes it possible to introduce a sequence of exogenous transfer efficiency. DNA (knock-in) at a specific site to prevent deleterious effects from random integration into the genome, which Acknowledgments is important for transgenic animal production (Hsu et al., 2014). FAPERJ grant 112.123/2012 and FAPESP grant A recent study produced cattle with increased 2015/26818-5. resistance to tuberculosis, by inserting natural resistance-associated macrophage protein-1 (NRAMP1), References using the single Cas9 nickase (Cas9n)-mediated single- strand break (SSB) for the first time, with the potential An LY, Yuan YG, Yu BL, Yang TJ, Cheng Y. 2012.

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